US6140066A - Methods of cancer diagnosis using a chimeric toxin - Google Patents

Methods of cancer diagnosis using a chimeric toxin Download PDF

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US6140066A
US6140066A US09/046,992 US4699298A US6140066A US 6140066 A US6140066 A US 6140066A US 4699298 A US4699298 A US 4699298A US 6140066 A US6140066 A US 6140066A
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adenocarcinoma
gly
ala
leu
chimeric toxin
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Haya Lorberboum-Galski
Shai Yarkoni
Ahmi Ben-Yehudah
Irina Marianovsky
Amotz Nechushtan
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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Assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM reassignment YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEN-YEHUDAH, AHMI, MECHUSHTAN, AMOTZ, YARKONI, SHAI, MARIANOVSKY, IRINA, LORBERBOUM-GALSKI, HAYA
Priority to NZ507294A priority patent/NZ507294A/en
Priority to DE69939819T priority patent/DE69939819D1/en
Priority to EP99910652A priority patent/EP1066390B1/en
Priority to CN99806580A priority patent/CN1303438A/en
Priority to AU29551/99A priority patent/AU758839B2/en
Priority to AT99910652T priority patent/ATE412756T1/en
Priority to CA002323786A priority patent/CA2323786A1/en
Priority to PCT/IL1999/000166 priority patent/WO1999049059A2/en
Priority to IL13865599A priority patent/IL138655A0/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Definitions

  • the present invention relates to methods for cancer diagnosis using a chimeric toxin.
  • the invention relates to the use of a chimeric toxin composed of gonadotropin releasing hormone (GnRH) and Pseudomonas exotoxin A (PE) to detect a tumor-associated epitope expressed by human adenocarcinomas.
  • GnRH gonadotropin releasing hormone
  • PE Pseudomonas exotoxin A
  • GnRH is a decapeptide produced by hypothalamic neurons and secreted into the hypophysioportal circulation via portal vessels. It is first synthesized as a larger precursor protein which is processed by proteolytic cleavage and amidation at its C-terminal glycine. GnRH stimulates gonadotroph cells in the anterior pituitary gland to release luteinizing hormone and follicle-stimulating hormone, thereby regulating the hypothalamic-pituitary gonadal control of human reproduction.
  • GnRH GnRH receptor
  • GnRH binding sites have been demonstrated in tumors, such tumors were derived mainly from hormone dependent tissues. Recently, Nechushtan et al. reported that certain hormone non-responsive tumors such as colon carcinomas, renal cell carcinomas and hepatocellular carcinomas were susceptible to killing by a chimeric toxin, GnRH-PE (J. Biol. Chem., 1997, 272:11597). GnRH caused the chimeric toxin to bind to GnRHR-expressing tumor cells, whereas PE mediated cell killing by inhibiting protein synthesis. However, prior to the present invention, it was not known whether the observed effects were due to the expression of a natural GnRHR by hormone non-responsive tumors or a new epitope recognized by GnRH-PE that was distinct from that bound by GnRH.
  • the present invention relates to methods for detecting a tumor cell using a GnRH-PE chimeric toxin, and GnRH-PE chimeric toxins that bind but do not kill tumor cells.
  • a GnRH-PE chimeric toxin to detect an epitope expressed by adenocarcinomas.
  • the GnRH-PE is modified to reduce its cytotoxic activities without altering its binding specificity to tumor cells.
  • Such molecules are particularly useful for the detection of tumor cells in a biological specimen and in a human subject who has cancer.
  • the invention is based, in part, on Applicants' discovery that two mutated recombinant chimeric toxins composed of GnRH and PE, referred to as LGnRH-PE40M and LGnRH-PE66M, bind to tumor cells without killing them. Since these chimeric toxins do not bind granulosa tumor cells which express natural GnRHR recognized by GnRH, the chimeric toxins of the invention recognize a new tumor-associated epitope expressed by adenocarcinomas.
  • FIGS. 1A, 1B and 1C Nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of LGnRH-PE66. Amino acid residue #575 identified within a square is deleted in a mutated chimeric toxin, LGnRH-PE66M.
  • FIG. 2 Nucleotide sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of LGnRH-PE40. Amino acid residue #336 identified within a square is deleted in a mutated chimeric toxin, LGnRH-PE40M.
  • FIG. 3 Mutated GnRH-PE chimeric toxins, LGnRH-PE40M and LGnRH-PE66M, did not exhibit ADP-ribosylation activities.
  • FIG. 4 Mutated GnRH-PE chimeric toxins, LGnRH-PE40M and LGnRH-PE66M, did not inhibit protein synthesis in 293 renal carcinoma cells, while the non-mutated chimeric toxins showed cytotoxic activities. Inhibition of protein synthesis is used as an indication of cytotoxicity.
  • FIG. 5 GnRH-PE chimeric toxins did not inhibit protein synthesis of primary cultures of granulosa tumor cells which expressed natural GnRHR.
  • GnRH-PE chimeric toxins of the present invention may be produced by chemical synthetic methods or by chemical linkage between the two moieties, it is preferred that they are produced by fusion of a coding sequence for GnRH and a coding sequence for PE under the control of a regulatory sequence which directs the expression of the fusion polynucleotide in an appropriate host cell (Nechushtan et al., 1997, J. Biol. Chem. 272:11597).
  • the fusion of two coding sequences can be achieved by methods well known in the art of molecular biology.
  • the PE coding sequence suitable for use in the present invention includes but is not limited to, full length PE, partial fragments of PE such as domains II and/or III of PE, mutated PE in which amino acid residues in domain I have been altered to reduce non-specific cytotoxicity and mutated PE which has minimal cytotoxic activities (U.S. Pat. No. 4,892,827, Lorberboum-Galski et al., 1990, J. Biol. Chem. 265:16311).
  • a fusion polynucleotide contain only the AUG translation initiation codon at the 5' end of the first coding sequence without the initiation codon of the second coding sequence to avoid the production of two encoded products.
  • a leader sequence may be placed at the 5' end of the polynucleotide in order to target the expressed product to a specific site or compartment within a host cell to facilitate secretion or subsequent purification after gene expression.
  • the two coding sequences can be fused directly without any linker or by using a flexible polylinker composed of the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO:5) repeated 1 to 3 times.
  • linker has been used in constructing single chain antibodies (scFv) by being inserted between V H and V L (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).
  • the linker is designed to enable the correct interaction between two beta-sheets forming the variable region of the single chain antibody.
  • Other linkers which may be used include Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:6) (Chaudhary et al., 1990, Proc. Natl.
  • a polynucleotide which encodes a GnRH-PE chimeric toxin, mutant polypeptides, biologically active fragments of chimeric protein, or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of the chimeric toxin, mutant polypeptides, peptide fragments, or a functional equivalent thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of the chimeric toxin.
  • Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent fusion gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within a chimeric sequence, which result in a silent change thus producing a functionally equivalent chimeric protein.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine, histidine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: glycine, asparagine, glutamine, serine, threonine, tyrosine; and amino acids with nonpolar head groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan.
  • DNA sequences of the invention may be engineered in order to alter a chimeric coding sequence for a variety of ends, including but not limited to, alterations which modify processing and expression of the gene product.
  • mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to reduce cytotoxicities, etc.
  • the coding sequence of the GnRH-PE chimeric toxin could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.
  • GnRH decapeptide and specific domains of PE can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography followed by chemical linkage to form a chimeric toxin (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).
  • the GnRH and PE produced by synthetic or recombinant methods may be conjugated by chemical linkers according to methods well known in the art (Brinkmann and Pastan, 1994, Biochemica et Biophysica Acta 1198:27-45).
  • the nucleotide sequence coding for a chimeric toxin, or a functional equivalent is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • the chimeric toxin as well as host cells or cell lines transfected or transformed with recombinant chimeric expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that bind to epitopes of the proteins to facilitate their purification.
  • a variety of host-expression vector systems may be utilized to express the GnRH-PE chimeric protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the chimeric toxin coding sequence; yeast transformed with recombinant yeast expression vectors containing the chimeric toxin coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the chimeric toxin coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the chimeric toxin coding sequence; or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA,
  • the chimeric toxins of the invention be expressed in prokaryotic or lower eukaryotic cells.
  • Section 6 illustrates that GnRH-PE chimeric toxins can be efficiently expressed in E. coli.
  • the mutated GnRH-PE chimeric toxins in Section 6, infra do not exhibit cytotoxic activities towards human cells, they may be expressed in eukaryotic cells as well.
  • each system vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage X, plac, ptrp, ptac (ptrp-lac hybrid promoter; cytomegalovirus promoter) and the like may be used;
  • promoters such as the baculovirus polyhedrin promoter may be used;
  • promoters derived from the genome of plant cells e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll ⁇ / ⁇ binding protein
  • plant viruses e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV
  • a number of expression vectors may be advantageously selected depending upon the use intended for the chimeric toxin expressed. For example, when large quantities of chimeric toxin are to be produced, vectors which direct the expression of high levels of protein products that are readily purified may be desirable. Such vectors include but are not limited to the pHL906 vector (Fishman et al., 1994, Biochem. 33:6235-6243), the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
  • An alternative expression system which could be used to express chimeric toxin is an insect system.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the chimeric toxin coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of the chimeric protein coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • Specific initiation signals may also be required for efficient translation of the inserted chimeric toxin coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire chimeric gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where the chimeric toxin coding sequence does not include its own initiation codon, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the chimeric protein coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the chimeric toxin.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the chimeric protein may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, and the like.
  • bacterial host cells or eukaryotic cell lines which stably express the chimeric toxins may be engineered.
  • host cells can be transformed with a chimeric coding sequence controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A. 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk - , hgprt - or aprt - cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. U.S.A. 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol.
  • hygro which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) genes. Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. U.S.A.
  • ODC ornithine decarboxylase
  • the GnRH-PE chimeric toxins of the invention can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.
  • art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.
  • the actual conditions used to purify each protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
  • any antibody which specifically binds GnRH, PE or a conformational epitope created by the fusion of GnRH and PE may be used.
  • various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with GnRH-PE chimeric toxin or a portion thereof.
  • the protein may be attached to a suitable carrier, such as bovine serum albumin (BSA), by means of a side chain functional group or linkers attached to a side chain functional group.
  • BSA bovine serum albumin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum bacilli Calmette-Guerin
  • Monoclonal antibodies to GnRH-PE may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975, Nature 256:495-497). In addition, techniques developed for the production of "chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.
  • the GnRH-PE chimeric toxins of the invention may be used to detect human tumors in vitro and in vivo. It is preferred that such toxins be mutated to abrogate their cytotoxic properties without affecting their binding specificity for tumor cells. Two examples of such GnRH-PE are illustrated in Section 6, infra.
  • the GnRH-PE chimeric toxins of the invention may be used to detect an epitope expressed by a wide variety of human adenocarcinomas, including but not limited to, colon adenocarcinoma, breast adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, endometrial adenocarcinoma, kidney adenocarcinoma, liver adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, cervical adenocarcinoma, gall bladder adenocarcinoma and pancreatic adenocarcinoma.
  • the chimeric toxins of the invention are particularly useful in differentiating adenocarcinomas from non-adenocarcinomas and normal cells that express the natural GnRHR.
  • the GnRH-PE chimeric toxins of the present invention can be used to detect cancer cells in a biological specimen such as histological and cytological specimens, and, in particular, to distinguish malignant tumors from normal tissues and non-malignant tumors for determination of surgical margin and an improved histological characterization of poorly differentiated tumors.
  • Tissue specimens may be stained by the chimeric toxins and their binding detected by a secondary antibody specific for a portion of the chimeric toxin.
  • the secondary antibody is conjugated to a detectable label such as a radioisotope, an enzyme such as peroxidase and alkaline phosphatase, an ultrasonic probe, a nuclear magnetic resonance (NMR) probe, and the like.
  • immunofluorescence techniques can use GnRH-PE to examine human tissue, cell and bodily fluid specimens.
  • slides containing cryostat sections of frozen, unfixed tissue biopsy samples or cytological smears are air dried, formalin or acetone fixed, and incubated with the GnRH-PE in a humidified chamber at room temperature.
  • the slides are then washed and further incubated with a preparation of a secondary antibody directed against GnRH-PE.
  • the secondary antibody is tagged with a compound such as rhodamine, phycoerythrin or fluorescein isothiocyanate, that fluoresces at a particular wavelength.
  • the staining pattern and intensities within the sample are then determined by fluorescent light microscopy and optionally photographically recorded.
  • computer enhanced fluorescence image analysis or flow cytometry can be used to examine tissue specimens or exfoliated cells, i.e., single cell preparations from aspiration biopsies of tumors using GnRH-PE.
  • the GnRH-PE chimeric toxins of the invention are particularly useful in quantitation of live tumor cells, i.e., single cell preparations from aspiration biopsies of adenocarcinomas by computer enhanced fluorescence image analyzer or with a flow cytometer.
  • the percent GnRH-PE-bound cell population, alone or in conjunction with determination of the DNA ploidy of these cells, may, additionally, provide very useful prognostic information by providing an early indicator of disease progression.
  • GnRH-PE can be extended to the screening of human biological fluids for the presence of the specific antigenic determinants recognized. In vitro immunoserological evaluation of biological fluids withdrawn from patients thereby permits non-invasive diagnosis of cancers.
  • human bodily fluids such as whole blood, pleural effusion fluid, cerebral spinal fluid, synovial fluid, prostatic fluid, seminal fluid or urine can be taken from a patient and assayed for the specific epitope, either as released antigen or membrane-bound on cells in the sample fluid, using GnRH-PE in standard radioimmunoassays or enzyme-linked immunoassays, competitive binding enzyme-linked immunoassays, dot blot or Western blot, or other assays known in the art.
  • Kits containing GnRH-PE can be prepared for in vitro diagnosis, prognosis and/or monitoring adenocarcinomas by the immunohistological, immunocytological and immunoserological methods described above.
  • the components of the kits can be packaged either in aqueous medium or in lyophilized form.
  • the GnRH-PE is used in the kits in the form of conjugates in which a label moiety is attached, such as an enzyme or a radioactive metal ion
  • the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
  • a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like.
  • a first of said container means or series of container means may contain GnRH-PE.
  • a second container means or series of container means may contain a label or linker-label intermediate capable of binding to GnRH-PE.
  • GnRH-PE chimeric toxins are also useful for targeting adenocarcinoma cells in vivo. They can be used for tumor localization in the detection and monitoring of primary tumors as well as metastases, especially lymph nodes. Primary evaluation of the extent of tumor spread may influence the choice of therapeutic modalities. Continued monitoring of residual tumors may also contribute to better surveillance and early initiation of salvage therapy. Tagged GnRH-PE may also be used intraoperatively for better debulking of a tumor, and minimizes normal tissue destruction such as lymph nodes. For these in vivo applications, it is preferred that highly purified GnRH-PE be used.
  • the purified GnRH-PE can be covalently attached, either directly or via a linker, to a compound which serves as a reporter group to permit imaging of specific tissues or organs following administration and localization of the conjugates or complexes.
  • a reporter group can serve as the reporter group, including such as radiopaque dyes, radioactive metal and non-metal isotopes, fluorogenic compounds, fluorescent compounds, positron emitting isotopes, non-paramagnetic metals, etc.
  • Kits for use with such in vivo tumor localization methods containing GnRH-PE (or fragments thereof) conjugated to any of the above types of substances can be prepared.
  • the components of the kits can be packaged either in aqueous medium or in lyophilized form.
  • the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
  • a plasmid vector carrying a full length PE gene (pJY3A1136-1,3) (Chaudhary et al., 1990, J. Biol. Chem. 265:16306-16310; Neshushtan et al., 1997, J. Biol. Chem. 272:11597) was cut with NdeI and HindIII.
  • a 36 base pair (bp) synthetic oligomer flanked by NdeI (5' end) and HindIII (3' end) restriction sites was ligated to the vector.
  • This oligomer insert contained a GnRH coding sequence in which the encoded amino acid at residue #6 was tryptophan instead of glycine.
  • the plasmid vector encoding LGnRH-PE66 was digested with NdeI and BamHI and ligated to a NdeI-BamHI 750 bp fragment obtained from the plasmid PHL-906 (Fishman et al., 1994, Biochemistry 33:6235-6243) along with the 36 bp synthetic oligomer consisting of the GnRH coding sequence with tryptophan replacing glycine at the sixth amino acid position.
  • a sequence encoding the above linker was again placed between the GnRH coding sequence and the PE coding sequence.
  • GnRH-PE chimeric toxins that were not cytotoxic to human cells
  • the region in the two aforementioned plasmids that encoded 122 amino acids at the C-terminal end of PE of LGnRH-PE66 and LGnRH-PE40 was excised with BamHI and EcoRI and replaced with a corresponding fragment which contained a deletion of a single codon encoding the amino acid at position 553 of the native PE molecule (FIGS. 1A, 1B and 2) (Fishman et al., 1997, Eur. J. Immunol. 27:486; Lukoc et al., 1988, Infect. Immun. 56:3095).
  • the mutated chimeric toxins are referred to as LGnRH-PE66M and LGnRH-PE40M, respectively.
  • the plasmids, pVM1 and pVM2, encoding the mutated GnRH-PE chimeric toxins, LGnRH-PE66M and LGnRH-PE40M, respectively, were expressed in E. coli strain BL21 (XDE3).
  • the plasmids that encoded LGnRH-PE40 and LGnRH-PE66 were also expressed in the same bacteria.
  • the plasmids were transferred into E. coli and the cells were grown in medium containing ampicillin. After reaching an A 600 value of 1.5-1.7, the cultures were induced at 37° C. with 1 mM isopropyl-1-thio- ⁇ -D-galactopyranoside. The cells were collected by centrifugation and the pellet was stored at -70° C. for several hours.
  • a pellet of expressing cells was suspended in lysis buffer (50 mM Tris-HCl at pH 8.0, 1 mM EDTA containing 0.2 mg/ml lysosyme), sonicated (three 30 second bursts) and centrifuged at 30,000 ⁇ g for 30 min. The supernatant (soluble fraction) was removed and kept for analysis. The pellet (insoluble fraction) was denatured in extraction buffer (6 M guanidinium-HCl, 0.1 M Tris-HCl, pH 8.6, 1 mM EDTA, 0.05 M NaCl, and 10 mM dithiothreitol) and stirred for 30 min at 4° C.
  • the suspension was cleared by centrifugation at 30000 ⁇ g for 15 min and the pellet discarded.
  • the supernatant was then dialyzed against 0.1 M Tris-HCl pH 8.0, 1 mM EDTA, 0.25 mM NaCl, and 0.25 mM L-arginine for 16 hours.
  • the dialyzate was centrifuged at 15000 ⁇ g for 15 min and the resulting supernatant (refolding fraction) was used as a source of the GnRH-PE chimeric toxins.
  • the refolded protein fractions were diluted with TE20 buffer (20 mM Tris, pH 8.0, 1 mM EDTA). DEAE Sepharose (Pharmacia, Sweden) was added and stirred for half an hour at 4° C. before being packed into a column. Washing of the column was done with 80 mM NaCl or 50 mM Nacl in TE20 buffer. Elution of protein was performed with the linear gradient of 2 ⁇ 200 ml of 0.08-0.35 M NaCl in TE20 (20 mM Tris pH 8.0, 1 mM EDTA) buffer. The peak fractions were pooled, dialyzed against phosphate saline buffer and kept in aliquots at -20° C.
  • a recombinant GnRH-PE chimeric toxin, LGnRH-PE66 was produced by fusion of a GnRH coding sequence and a PE coding sequence with the insertion of a linker between the two moieties.
  • a second GnRH-PE chimeric toxin, LGnRH-PE40 was produced in a similar manner except that only domains II and III of PE was encoded by the toxin coding sequence.
  • the coding sequences of these two chimeric toxins were mutated to result in a single amino acid deletion in the PE portion.
  • the mutated chimeric toxins were also expressed as recombinant proteins.
  • the four GnRH-PE chimeric toxins were purified from E. coli lysates and refolded. Since PE kills eukaryotic cells by inactivating elongation factor 2 through ADP-ribosylation during protein synthesis, the four forms of GnRH-PE chimeric toxins were tested in a cell free assay for their enzymatic activities in ADP-ribosylation (Chung and Collier, 1977, J. Infect. Immun. 16:832-841).

Abstract

The present invention relates to methods for cancer diagnosis using a chimeric toxin. In particular, the invention relates to the use of a chimeric toxin composed of gonadotropin releasing hormone (GnRH) and Pseudomonas exotoxin A (PE) to detect a tumor-associated epitope expressed by human adenocarcinomas. Mutated GnRH-PE molecules that bind but do not kill tumor cells are exemplified.

Description

INTRODUCTION
The present invention relates to methods for cancer diagnosis using a chimeric toxin. In particular, the invention relates to the use of a chimeric toxin composed of gonadotropin releasing hormone (GnRH) and Pseudomonas exotoxin A (PE) to detect a tumor-associated epitope expressed by human adenocarcinomas. Mutated GnRH-PE molecules that bind but do not kill tumor cells are exemplified.
BACKGROUND OF THE INVENTION
GnRH is a decapeptide produced by hypothalamic neurons and secreted into the hypophysioportal circulation via portal vessels. It is first synthesized as a larger precursor protein which is processed by proteolytic cleavage and amidation at its C-terminal glycine. GnRH stimulates gonadotroph cells in the anterior pituitary gland to release luteinizing hormone and follicle-stimulating hormone, thereby regulating the hypothalamic-pituitary gonadal control of human reproduction.
The involvement of GnRH has been implicated in certain carcinomas, and GnRH analogues have been used in the treatment of breast, prostatic, pancreatic, endometrial and ovarian cancers (Kadar et al., 1988, Prostate 12:229-307). The analogues suppressed tumor cell growth in vitro and in vivo. In addition, GnRH binding sites have been reported in certain solid tumors and in established cell lines (Emons et al., 1993, J. Clin. Endocrinol. Metab. 77:1458-1464), though preliminary results suggest that the GnRH receptor (GnRHR) involved might differ from the previously documented receptor (Kadar et al., 1992, Biochem. Biophs. Res. Comm. 189:289-295).
Although GnRH binding sites have been demonstrated in tumors, such tumors were derived mainly from hormone dependent tissues. Recently, Nechushtan et al. reported that certain hormone non-responsive tumors such as colon carcinomas, renal cell carcinomas and hepatocellular carcinomas were susceptible to killing by a chimeric toxin, GnRH-PE (J. Biol. Chem., 1997, 272:11597). GnRH caused the chimeric toxin to bind to GnRHR-expressing tumor cells, whereas PE mediated cell killing by inhibiting protein synthesis. However, prior to the present invention, it was not known whether the observed effects were due to the expression of a natural GnRHR by hormone non-responsive tumors or a new epitope recognized by GnRH-PE that was distinct from that bound by GnRH.
SUMMARY OF THE INVENTION
The present invention relates to methods for detecting a tumor cell using a GnRH-PE chimeric toxin, and GnRH-PE chimeric toxins that bind but do not kill tumor cells. In particular, it relates to the use of a GnRH-PE chimeric toxin to detect an epitope expressed by adenocarcinomas. For the practice of the invention, it is preferred that the GnRH-PE is modified to reduce its cytotoxic activities without altering its binding specificity to tumor cells. Such molecules are particularly useful for the detection of tumor cells in a biological specimen and in a human subject who has cancer.
The invention is based, in part, on Applicants' discovery that two mutated recombinant chimeric toxins composed of GnRH and PE, referred to as LGnRH-PE40M and LGnRH-PE66M, bind to tumor cells without killing them. Since these chimeric toxins do not bind granulosa tumor cells which express natural GnRHR recognized by GnRH, the chimeric toxins of the invention recognize a new tumor-associated epitope expressed by adenocarcinomas.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B and 1C. Nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of LGnRH-PE66. Amino acid residue #575 identified within a square is deleted in a mutated chimeric toxin, LGnRH-PE66M.
FIG. 2. Nucleotide sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of LGnRH-PE40. Amino acid residue #336 identified within a square is deleted in a mutated chimeric toxin, LGnRH-PE40M.
FIG. 3. Mutated GnRH-PE chimeric toxins, LGnRH-PE40M and LGnRH-PE66M, did not exhibit ADP-ribosylation activities.
FIG. 4. Mutated GnRH-PE chimeric toxins, LGnRH-PE40M and LGnRH-PE66M, did not inhibit protein synthesis in 293 renal carcinoma cells, while the non-mutated chimeric toxins showed cytotoxic activities. Inhibition of protein synthesis is used as an indication of cytotoxicity.
FIG. 5. GnRH-PE chimeric toxins did not inhibit protein synthesis of primary cultures of granulosa tumor cells which expressed natural GnRHR.
DETAILED DESCRIPTION OF THE INVENTION Production of GnRH-PE Chimeric Toxins
While the GnRH-PE chimeric toxins of the present invention may be produced by chemical synthetic methods or by chemical linkage between the two moieties, it is preferred that they are produced by fusion of a coding sequence for GnRH and a coding sequence for PE under the control of a regulatory sequence which directs the expression of the fusion polynucleotide in an appropriate host cell (Nechushtan et al., 1997, J. Biol. Chem. 272:11597). The fusion of two coding sequences can be achieved by methods well known in the art of molecular biology. The PE coding sequence suitable for use in the present invention, includes but is not limited to, full length PE, partial fragments of PE such as domains II and/or III of PE, mutated PE in which amino acid residues in domain I have been altered to reduce non-specific cytotoxicity and mutated PE which has minimal cytotoxic activities (U.S. Pat. No. 4,892,827, Lorberboum-Galski et al., 1990, J. Biol. Chem. 265:16311).
It is preferred that a fusion polynucleotide contain only the AUG translation initiation codon at the 5' end of the first coding sequence without the initiation codon of the second coding sequence to avoid the production of two encoded products. In addition, a leader sequence may be placed at the 5' end of the polynucleotide in order to target the expressed product to a specific site or compartment within a host cell to facilitate secretion or subsequent purification after gene expression. The two coding sequences can be fused directly without any linker or by using a flexible polylinker composed of the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO:5) repeated 1 to 3 times. Such linker has been used in constructing single chain antibodies (scFv) by being inserted between VH and VL (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883). The linker is designed to enable the correct interaction between two beta-sheets forming the variable region of the single chain antibody. Other linkers which may be used include Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:6) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (SEQ ID NO:7) (Bird et al., 1988, Science 242:423-426).
Expression of GnRH-PE Chimeric Toxins
A polynucleotide which encodes a GnRH-PE chimeric toxin, mutant polypeptides, biologically active fragments of chimeric protein, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of the chimeric toxin, mutant polypeptides, peptide fragments, or a functional equivalent thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of the chimeric toxin.
Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent fusion gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within a chimeric sequence, which result in a silent change thus producing a functionally equivalent chimeric protein. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine, histidine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: glycine, asparagine, glutamine, serine, threonine, tyrosine; and amino acids with nonpolar head groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan.
The DNA sequences of the invention may be engineered in order to alter a chimeric coding sequence for a variety of ends, including but not limited to, alterations which modify processing and expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to reduce cytotoxicities, etc.
In an alternate embodiment of the invention, the coding sequence of the GnRH-PE chimeric toxin could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817. In addition, GnRH decapeptide and specific domains of PE can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography followed by chemical linkage to form a chimeric toxin (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49). Alternatively, the GnRH and PE produced by synthetic or recombinant methods may be conjugated by chemical linkers according to methods well known in the art (Brinkmann and Pastan, 1994, Biochemica et Biophysica Acta 1198:27-45).
In order to express a biologically active GnRH-PE chimeric toxin, the nucleotide sequence coding for a chimeric toxin, or a functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The chimeric toxin as well as host cells or cell lines transfected or transformed with recombinant chimeric expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that bind to epitopes of the proteins to facilitate their purification.
Methods which are well known to those skilled in the art can be used to construct expression vectors containing the GnRH-PE chimeric toxin coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.
A variety of host-expression vector systems may be utilized to express the GnRH-PE chimeric protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the chimeric toxin coding sequence; yeast transformed with recombinant yeast expression vectors containing the chimeric toxin coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the chimeric toxin coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the chimeric toxin coding sequence; or animal cell systems. It should be noted that since PE normally kills mammalian cells, it is preferred that the chimeric toxins of the invention be expressed in prokaryotic or lower eukaryotic cells. Section 6 illustrates that GnRH-PE chimeric toxins can be efficiently expressed in E. coli. However, since the mutated GnRH-PE chimeric toxins in Section 6, infra, do not exhibit cytotoxic activities towards human cells, they may be expressed in eukaryotic cells as well.
The expression elements of each system vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage X, plac, ptrp, ptac (ptrp-lac hybrid promoter; cytomegalovirus promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll α/β binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the chimeric DNA, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the chimeric toxin expressed. For example, when large quantities of chimeric toxin are to be produced, vectors which direct the expression of high levels of protein products that are readily purified may be desirable. Such vectors include but are not limited to the pHL906 vector (Fishman et al., 1994, Biochem. 33:6235-6243), the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the chimeric protein coding sequence may be ligated into the vector in frame with the lacZ coding region so that a hybrid lacZ protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.
An alternative expression system which could be used to express chimeric toxin is an insect system. In one such system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The chimeric toxin coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the chimeric protein coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g., see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).
Specific initiation signals may also be required for efficient translation of the inserted chimeric toxin coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire chimeric gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where the chimeric toxin coding sequence does not include its own initiation codon, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the chimeric protein coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the chimeric toxin. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the chimeric protein may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, and the like.
For long-term, high-yield production of recombinant chimeric toxins, stable expression is preferred. For example, bacterial host cells or eukaryotic cell lines which stably express the chimeric toxins may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with a chimeric coding sequence controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A. 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk- , hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. U.S.A. 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) genes. Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8047); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).
Protein Purification
The GnRH-PE chimeric toxins of the invention can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify each protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
For affinity chromatography purification, any antibody which specifically binds GnRH, PE or a conformational epitope created by the fusion of GnRH and PE may be used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with GnRH-PE chimeric toxin or a portion thereof. The protein may be attached to a suitable carrier, such as bovine serum albumin (BSA), by means of a side chain functional group or linkers attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
Monoclonal antibodies to GnRH-PE may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975, Nature 256:495-497). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce GnRH-PE-specific single chain antibodies for protein purification and detection.
Cancer Diagnosis Using GnRH-PE Chimeric Toxins
The GnRH-PE chimeric toxins of the invention may be used to detect human tumors in vitro and in vivo. It is preferred that such toxins be mutated to abrogate their cytotoxic properties without affecting their binding specificity for tumor cells. Two examples of such GnRH-PE are illustrated in Section 6, infra. The GnRH-PE chimeric toxins of the invention may be used to detect an epitope expressed by a wide variety of human adenocarcinomas, including but not limited to, colon adenocarcinoma, breast adenocarcinoma, lung adenocarcinoma, ovarian adenocarcinoma, endometrial adenocarcinoma, kidney adenocarcinoma, liver adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, cervical adenocarcinoma, gall bladder adenocarcinoma and pancreatic adenocarcinoma. The chimeric toxins of the invention are particularly useful in differentiating adenocarcinomas from non-adenocarcinomas and normal cells that express the natural GnRHR.
In Vitro Diagnostic Applications
The GnRH-PE chimeric toxins of the present invention can be used to detect cancer cells in a biological specimen such as histological and cytological specimens, and, in particular, to distinguish malignant tumors from normal tissues and non-malignant tumors for determination of surgical margin and an improved histological characterization of poorly differentiated tumors. Tissue specimens may be stained by the chimeric toxins and their binding detected by a secondary antibody specific for a portion of the chimeric toxin. The secondary antibody is conjugated to a detectable label such as a radioisotope, an enzyme such as peroxidase and alkaline phosphatase, an ultrasonic probe, a nuclear magnetic resonance (NMR) probe, and the like.
In addition, immunofluorescence techniques can use GnRH-PE to examine human tissue, cell and bodily fluid specimens. In a typical protocol, slides containing cryostat sections of frozen, unfixed tissue biopsy samples or cytological smears are air dried, formalin or acetone fixed, and incubated with the GnRH-PE in a humidified chamber at room temperature.
The slides are then washed and further incubated with a preparation of a secondary antibody directed against GnRH-PE. The secondary antibody is tagged with a compound such as rhodamine, phycoerythrin or fluorescein isothiocyanate, that fluoresces at a particular wavelength. The staining pattern and intensities within the sample are then determined by fluorescent light microscopy and optionally photographically recorded.
In another embodiment, computer enhanced fluorescence image analysis or flow cytometry can be used to examine tissue specimens or exfoliated cells, i.e., single cell preparations from aspiration biopsies of tumors using GnRH-PE. The GnRH-PE chimeric toxins of the invention are particularly useful in quantitation of live tumor cells, i.e., single cell preparations from aspiration biopsies of adenocarcinomas by computer enhanced fluorescence image analyzer or with a flow cytometer. The percent GnRH-PE-bound cell population, alone or in conjunction with determination of the DNA ploidy of these cells, may, additionally, provide very useful prognostic information by providing an early indicator of disease progression.
The use of GnRH-PE can be extended to the screening of human biological fluids for the presence of the specific antigenic determinants recognized. In vitro immunoserological evaluation of biological fluids withdrawn from patients thereby permits non-invasive diagnosis of cancers. By way of illustration, human bodily fluids such as whole blood, pleural effusion fluid, cerebral spinal fluid, synovial fluid, prostatic fluid, seminal fluid or urine can be taken from a patient and assayed for the specific epitope, either as released antigen or membrane-bound on cells in the sample fluid, using GnRH-PE in standard radioimmunoassays or enzyme-linked immunoassays, competitive binding enzyme-linked immunoassays, dot blot or Western blot, or other assays known in the art.
Kits containing GnRH-PE can be prepared for in vitro diagnosis, prognosis and/or monitoring adenocarcinomas by the immunohistological, immunocytological and immunoserological methods described above. The components of the kits can be packaged either in aqueous medium or in lyophilized form. When the GnRH-PE is used in the kits in the form of conjugates in which a label moiety is attached, such as an enzyme or a radioactive metal ion, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
A kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like. A first of said container means or series of container means may contain GnRH-PE. A second container means or series of container means may contain a label or linker-label intermediate capable of binding to GnRH-PE.
In Vivo Diagnostic Applications
GnRH-PE chimeric toxins are also useful for targeting adenocarcinoma cells in vivo. They can be used for tumor localization in the detection and monitoring of primary tumors as well as metastases, especially lymph nodes. Primary evaluation of the extent of tumor spread may influence the choice of therapeutic modalities. Continued monitoring of residual tumors may also contribute to better surveillance and early initiation of salvage therapy. Tagged GnRH-PE may also be used intraoperatively for better debulking of a tumor, and minimizes normal tissue destruction such as lymph nodes. For these in vivo applications, it is preferred that highly purified GnRH-PE be used.
For in vivo detection and/or monitoring of adenocarcinomas, the purified GnRH-PE can be covalently attached, either directly or via a linker, to a compound which serves as a reporter group to permit imaging of specific tissues or organs following administration and localization of the conjugates or complexes. A variety of different types of substances can serve as the reporter group, including such as radiopaque dyes, radioactive metal and non-metal isotopes, fluorogenic compounds, fluorescent compounds, positron emitting isotopes, non-paramagnetic metals, etc.
Kits for use with such in vivo tumor localization methods containing GnRH-PE (or fragments thereof) conjugated to any of the above types of substances can be prepared. The components of the kits can be packaged either in aqueous medium or in lyophilized form. When the chimeric toxins are used in the kits in the form of conjugates in which a label is attached, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates or as separate moieties to be conjugated by the user of the kit.
EXAMPLE Mutated GnRH-PE Chimeric Toxins Bound but did not Kill Tumor Cells Materials and Methods
Construction of GnRH-PE Chimeric Toxins
A plasmid vector carrying a full length PE gene (pJY3A1136-1,3) (Chaudhary et al., 1990, J. Biol. Chem. 265:16306-16310; Neshushtan et al., 1997, J. Biol. Chem. 272:11597) was cut with NdeI and HindIII. A 36 base pair (bp) synthetic oligomer flanked by NdeI (5' end) and HindIII (3' end) restriction sites was ligated to the vector. This oligomer insert contained a GnRH coding sequence in which the encoded amino acid at residue #6 was tryptophan instead of glycine. In addition, a sequence encoding a linker Gly-Gly-Gly-Gly-Ser (SEQ ID NO:5) repeated twice was placed between the GnRH coding sequence and the PE coding sequence. The resultant plasmid encoded a chimeric toxin, LGnRH-PE66, and it was confirmed by restriction endonuclease digestion and DNA sequence analysis (FIGS. 1A and 1B).
In order to produce a second chimeric toxin, LGnRH-PE40, the plasmid vector encoding LGnRH-PE66 was digested with NdeI and BamHI and ligated to a NdeI-BamHI 750 bp fragment obtained from the plasmid PHL-906 (Fishman et al., 1994, Biochemistry 33:6235-6243) along with the 36 bp synthetic oligomer consisting of the GnRH coding sequence with tryptophan replacing glycine at the sixth amino acid position. A sequence encoding the above linker was again placed between the GnRH coding sequence and the PE coding sequence. The resultant plasmid encoded a chimeric toxin, LGnRH-PE40, and it was confirmed by restriction endonuclease digestion and DNA sequence analysis (FIG. 2). The toxin encoded by this plasmid consisted of domains II and III of the full-length PE.
Generation of Mutated GnRH-PE Chimeric Toxins
In order to construct GnRH-PE chimeric toxins that were not cytotoxic to human cells, the region in the two aforementioned plasmids that encoded 122 amino acids at the C-terminal end of PE of LGnRH-PE66 and LGnRH-PE40 was excised with BamHI and EcoRI and replaced with a corresponding fragment which contained a deletion of a single codon encoding the amino acid at position 553 of the native PE molecule (FIGS. 1A, 1B and 2) (Fishman et al., 1997, Eur. J. Immunol. 27:486; Lukoc et al., 1988, Infect. Immun. 56:3095). The mutated chimeric toxins are referred to as LGnRH-PE66M and LGnRH-PE40M, respectively.
Expression of GnRH-PE Chimeric Toxins
The plasmids, pVM1 and pVM2, encoding the mutated GnRH-PE chimeric toxins, LGnRH-PE66M and LGnRH-PE40M, respectively, were expressed in E. coli strain BL21 (XDE3). The plasmids that encoded LGnRH-PE40 and LGnRH-PE66 were also expressed in the same bacteria. The plasmids were transferred into E. coli and the cells were grown in medium containing ampicillin. After reaching an A600 value of 1.5-1.7, the cultures were induced at 37° C. with 1 mM isopropyl-1-thio-β-D-galactopyranoside. The cells were collected by centrifugation and the pellet was stored at -70° C. for several hours.
A pellet of expressing cells was suspended in lysis buffer (50 mM Tris-HCl at pH 8.0, 1 mM EDTA containing 0.2 mg/ml lysosyme), sonicated (three 30 second bursts) and centrifuged at 30,000×g for 30 min. The supernatant (soluble fraction) was removed and kept for analysis. The pellet (insoluble fraction) was denatured in extraction buffer (6 M guanidinium-HCl, 0.1 M Tris-HCl, pH 8.6, 1 mM EDTA, 0.05 M NaCl, and 10 mM dithiothreitol) and stirred for 30 min at 4° C. The suspension was cleared by centrifugation at 30000×g for 15 min and the pellet discarded. The supernatant was then dialyzed against 0.1 M Tris-HCl pH 8.0, 1 mM EDTA, 0.25 mM NaCl, and 0.25 mM L-arginine for 16 hours. The dialyzate was centrifuged at 15000×g for 15 min and the resulting supernatant (refolding fraction) was used as a source of the GnRH-PE chimeric toxins.
Analysis of the fraction by SDS/PAGE revealed a major band corresponding to the chimeric toxin. Immunoblotting with polyclonal antibodies against PE confirmed the production of GnRH-PE chimeric toxins.
Purification of Recombinant GnRH-PE Chimeric Toxins
The refolded protein fractions were diluted with TE20 buffer (20 mM Tris, pH 8.0, 1 mM EDTA). DEAE Sepharose (Pharmacia, Sweden) was added and stirred for half an hour at 4° C. before being packed into a column. Washing of the column was done with 80 mM NaCl or 50 mM Nacl in TE20 buffer. Elution of protein was performed with the linear gradient of 2×200 ml of 0.08-0.35 M NaCl in TE20 (20 mM Tris pH 8.0, 1 mM EDTA) buffer. The peak fractions were pooled, dialyzed against phosphate saline buffer and kept in aliquots at -20° C.
Results
A recombinant GnRH-PE chimeric toxin, LGnRH-PE66, was produced by fusion of a GnRH coding sequence and a PE coding sequence with the insertion of a linker between the two moieties. A second GnRH-PE chimeric toxin, LGnRH-PE40, was produced in a similar manner except that only domains II and III of PE was encoded by the toxin coding sequence. In addition, the coding sequences of these two chimeric toxins were mutated to result in a single amino acid deletion in the PE portion. The mutated chimeric toxins were also expressed as recombinant proteins.
The four GnRH-PE chimeric toxins were purified from E. coli lysates and refolded. Since PE kills eukaryotic cells by inactivating elongation factor 2 through ADP-ribosylation during protein synthesis, the four forms of GnRH-PE chimeric toxins were tested in a cell free assay for their enzymatic activities in ADP-ribosylation (Chung and Collier, 1977, J. Infect. Immun. 16:832-841). While the two non-mutated GnRH-PE chimeric toxins, LGnRH-PE40 and LGnRH-PE66, exhibited ADP-ribosylation activities, the mutated chimeric toxins, LGnRH-PE40M and LGnRH-PE66M, were completely inactive in the same assay (FIG. 3). Thus, a single amino acid substitution in PE abrogated the enzymatic activities of the chimeric toxins.
In addition, all four GnRH-PE chimeric toxins were tested for their ability to kill 293 renal adenocarcinoma cells. It was shown that only the non-mutated chimeric toxins showed dose-dependent inhibition of protein synthesis in the target cells (FIG. 4). However, when the chimeric toxins were incubated with the same target cells and their binding was detected by a labeled anti-PE antibody and FACS analysis, all four toxins were able to bind renal carcinoma cells with no binding to control T24A bladder carcinoma cells. Therefore, while the mutated GnRH-PE chimeric toxins were not able to kill target cells, they retained the ability to bind to tumor cells. Such non-cytotoxic chimeric toxins are particularly useful for use in cancer diagnosis in vitro and in vivo.
Primary granulosa tumor cells were obtained and shown to express GnRHR by PCR using primers corresponding to specific portions of the GnRHR. The PCR product in granulosa cells was the same size as that obtained from pituitary cells which expressed natural GnRHR. In contrast, GnRHR-negative cells such as normal human lymphocytes did not produce a detectable PCR product. Notwithstanding their expression of natural GnRHR, the granulosa cells were not susceptible to killing by any of the four GnRH-PE chimeric toxins, indicating that the chimeric toxins bind to a new epitope expressed by adenocarcinoma cells that is distinct from that bound by GnRH itself (FIG. 5).
The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention and any sequences which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
All publications cited herein are incorporated by reference in their entirety.
__________________________________________________________________________
#             SEQUENCE LISTING                                            
   - -  - - (1) GENERAL INFORMATION:                                      
   - -    (iii) NUMBER OF SEQUENCES: 7                                    
   - -  - - (2) INFORMATION FOR SEQ ID NO:1:                              
   - -      (i) SEQUENCE CHARACTERISTICS:                                 
            (A) LENGTH: 1908 base - #pairs                                
            (B) TYPE: nucleic acid                                        
            (C) STRANDEDNESS: single                                      
            (D) TOPOLOGY: linear                                          
   - -     (ix) FEATURE:                                                  
            (A) NAME/KEY: Coding Se - #quence                             
            (B) LOCATION: 1...1905                                        
            (D) OTHER INFORMATION:                                        
   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                        
   - - ATG GAG CAC TGG TCC TAT TGG CTG CGC CCT GG - #A GAA GCT GGA GGA    
GGA       48                                                              
  Met Glu His Trp Ser Tyr Trp Leu Arg Pro Gl - #y Glu Ala Gly Gly Gly     
  1               5  - #                10  - #                15         
  - - GGA TCC GGA GGA GGA GGA TCC GGT CAA GCT TT - #C GAC CTC TGG AAC GAA 
      96                                                                  
 Gly Ser Gly Gly Gly Gly Ser Gly Gln Ala Ph - #e Asp Leu Trp Asn Glu      
             20      - #            25      - #            30             
  - - TGC GCC AAA GCC TGC GTG CTC GAC CTC AAG GA - #C GGC GTG CGT TCC AGC 
     144                                                                  
 Cys Ala Lys Ala Cys Val Leu Asp Leu Lys As - #p Gly Val Arg Ser Ser      
         35          - #        40          - #        45                 
  - - CGC ATG AGC GTC GAC CCG GCC ATC GCC GAC AC - #C AAC GGC CAG GGC GTG 
     192                                                                  
 Arg Met Ser Val Asp Pro Ala Ile Ala Asp Th - #r Asn Gly Gln Gly Val      
     50              - #    55              - #    60                     
  - - CTG CAC TAC TCC ATG GTC CTG GAG GGC GGC AA - #C GAC GCG CTC GAG CTG 
     240                                                                  
 Leu His Tyr Ser Met Val Leu Glu Gly Gly As - #n Asp Ala Leu Glu Leu      
 65                  - #70                  - #75                  - #80  
  - - GCC ATC GAC AAC GCC CTC AGC ATC ACC AGC GA - #C GGC CTG ACC ATC CGC 
     288                                                                  
 Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser As - #p Gly Leu Thr Ile Arg      
                 85  - #                90  - #                95         
  - - CTC GAA GGC GGC GTC GAG CCG AAC AAG CCG CT - #G CGC TAC AGC TAC ACG 
     336                                                                  
 Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Le - #u Arg Tyr Ser Tyr Thr      
             100      - #           105      - #           110            
  - - CGC CAG GCG CGC GGC AGG TGG TCG CTG AAC TG - #G CTG GTA CCG ATC GGC 
     384                                                                  
 Arg Gln Ala Arg Gly Arg Trp Ser Leu Asn Tr - #p Leu Val Pro Ile Gly      
         115          - #       120          - #       125                
  - - CAC GAG AAG CCC TCG AAC ATC AAG GTG TTC AT - #C CAC GAA CTG AAC GCC 
     432                                                                  
 His Glu Lys Pro Ser Asn Ile Lys Val Phe Il - #e His Glu Leu Asn Ala      
     130              - #   135              - #   140                    
  - - GGC AAC CAG CTC AGC CAC ATG TCG CCG ATC TA - #C ACC ATC GAG ATG GGC 
     480                                                                  
 Gly Asn Gln Leu Ser His Met Ser Pro Ile Ty - #r Thr Ile Glu Met Gly      
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - GAC GAG TTG CTG GCG AAG CTG GCG CGC GAT GC - #C ACC TTC TTC GTC    
AGG      528                                                              
  Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Al - #a Thr Phe Phe Val Arg     
                 165  - #               170  - #               175        
  - - GCG CAC GAG AGC AAC GAG ATG CAG CCG ACG CT - #C GCC ATC AGC CAT GCC 
     576                                                                  
 Ala His Glu Ser Asn Glu Met Gln Pro Thr Le - #u Ala Ile Ser His Ala      
             180      - #           185      - #           190            
  - - GGG GTC AGC GTG GTC ATG GCC CAG AAC CAG CC - #G CGC CGG GAA AAG CGC 
     624                                                                  
 Gly Val Ser Val Val Met Ala Gln Asn Gln Pr - #o Arg Arg Glu Lys Arg      
         195          - #       200          - #       205                
  - - TGG AGC GAA TGG GCC AGC GGC AAG GTG TTG TG - #C CTG CTC GAC CCG CTG 
     672                                                                  
 Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cy - #s Leu Leu Asp Pro Leu      
     210              - #   215              - #   220                    
  - - GAC GGG GTC TAC AAC TAC CTC GCC CAG CAA CG - #C TGC AAC CTC GAC GAT 
     720                                                                  
 Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Ar - #g Cys Asn Leu Asp Asp      
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - ACC TGG GAA GGC AAG ATC TAC CGG GTG CTC GC - #C GGC AAC CCG GCG    
AAG      768                                                              
  Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Al - #a Gly Asn Pro Ala Lys     
                 245  - #               250  - #               255        
  - - CAT GAC CTG GAC ATC AAA CCC ACG GTC ATC AG - #T GAA GAG CTG GAG TTT 
     816                                                                  
 His Asp Leu Asp Ile Lys Pro Thr Val Ile Se - #r Glu Glu Leu Glu Phe      
             260      - #           265      - #           270            
  - - CCC GAG GGC GGC AGC CTG GCC GCG CTG ACC GC - #G CAC CAG GCT TGC CAC 
     864                                                                  
 Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Al - #a His Gln Ala Cys His      
         275          - #       280          - #       285                
  - - CTG CCG CTG GAG ACT TTC ACC CGT CAT CGC CA - #G CCG CGC GGC TGG GAA 
     912                                                                  
 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gl - #n Pro Arg Gly Trp Glu      
     290              - #   295              - #   300                    
  - - CAA CTG GAG CAG TGC GGC TAT CCG GTG CAG CG - #G CTG GTC GCC CTC TAC 
     960                                                                  
 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Ar - #g Leu Val Ala Leu Tyr      
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - CTG GCG GCG CGG CTG TCG TGG AAC CAG GTC GA - #C CAG GTG ATC CGC    
AAC     1008                                                              
  Leu Ala Ala Arg Leu Ser Trp Asn Gln Val As - #p Gln Val Ile Arg Asn     
                 325  - #               330  - #               335        
  - - GCC CTG GCC AGC CCC GGC AGC GGC GGC GAC CT - #G GGC GAA GCG ATC CGC 
    1056                                                                  
 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Le - #u Gly Glu Ala Ile Arg      
             340      - #           345      - #           350            
  - - GAG CAG CCG GAG CAG GCC CGT CTG GCC CTG AC - #C CTG GCC GCC GCC GAG 
    1104                                                                  
 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Th - #r Leu Ala Ala Ala Glu      
         355          - #       360          - #       365                
  - - AGC GAG CGC TTC GTC CGG CAG GGC ACC GGC AA - #C GAC GAG GCC GGC GCG 
    1152                                                                  
 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly As - #n Asp Glu Ala Gly Ala      
     370              - #   375              - #   380                    
  - - GCC AAC GCC GAC GTG GTG AGC CTG ACC TGC CC - #G GTC GCC GCC GGT GAA 
    1200                                                                  
 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pr - #o Val Ala Ala Gly Glu      
 385                 3 - #90                 3 - #95                 4 -  
#00                                                                       
   - - TGC GCG GGC CCG GCG GAC AGC GGC GAC GCC CT - #G CTG GAG GCG AAC    
TAT     1248                                                              
  Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Le - #u Leu Glu Ala Asn Tyr     
                 405  - #               410  - #               415        
  - - CCC ACT GGC GCG GAG TTC CTC GGC GAC GGC GG - #C GAC GTC AGC TTC AGC 
    1296                                                                  
 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gl - #y Asp Val Ser Phe Ser      
             420      - #           425      - #           430            
  - - ACC CGC GGC ACG CAG AAC TGG ACG GTG GAG CG - #G CTG CTC CAG GCG CAC 
    1344                                                                  
 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Ar - #g Leu Leu Gln Ala His      
         435          - #       440          - #       445                
  - - CGC CAA CTG GAG GAG CGC GGC TAT GTG TTC GT - #C GGC TAC CAC GGC ACC 
    1392                                                                  
 Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Va - #l Gly Tyr His Gly Thr      
     450              - #   455              - #   460                    
  - - TTC CTC GAA GCG GCG CAA AGC ATC GTC TTC GG - #C GGG GTG CGC GCG CGC 
    1440                                                                  
 Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gl - #y Gly Val Arg Ala Arg      
 465                 4 - #70                 4 - #75                 4 -  
#80                                                                       
   - - AGC CAG GAC CTC GAC GCG ATC TGG CGC GGT TT - #C TAT ATC GCC GGC    
GAT     1488                                                              
  Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Ph - #e Tyr Ile Ala Gly Asp     
                 485  - #               490  - #               495        
  - - CCG GCG CTG GCC TAC GGC TAC GCC CAG GAC CA - #G GAA CCC GAC GCA CGC 
    1536                                                                  
 Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gl - #n Glu Pro Asp Ala Arg      
             500      - #           505      - #           510            
  - - GGC CGG ATC CGC AAC GGT GCC CTG CTG CGG GT - #C TAT GTG CCG CGC TCG 
    1584                                                                  
 Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Va - #l Tyr Val Pro Arg Ser      
         515          - #       520          - #       525                
  - - AGC CTG CCG GGC TTC TAC CGC ACC AGC CTG AC - #C CTG GCC GCG CCG GAG 
    1632                                                                  
 Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Th - #r Leu Ala Ala Pro Glu      
     530              - #   535              - #   540                    
  - - GCG GCG GGC GAG GTC GAA CGG CTG ATC GGC CA - #T CCG CTG CCG CTG CGC 
    1680                                                                  
 Ala Ala Gly Glu Val Glu Arg Leu Ile Gly Hi - #s Pro Leu Pro Leu Arg      
 545                 5 - #50                 5 - #55                 5 -  
#60                                                                       
   - - CTG GAC GCC ATC ACC GGC CCC GAG GAG GAA GG - #C GGG CGC CTG GAG    
ACC     1728                                                              
  Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gl - #y Gly Arg Leu Glu Thr     
                 565  - #               570  - #               575        
  - - ATT CTC GGC TGG CCG CTG GCC GAG CGC ACC GT - #G GTG ATT CCC TCG GCG 
    1776                                                                  
 Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Va - #l Val Ile Pro Ser Ala      
             580      - #           585      - #           590            
  - - ATC CCC ACC GAC CCG CGC AAC GTC GGC GGC GA - #C CTC GAC CCG TCC AGC 
    1824                                                                  
 Ile Pro Thr Asp Pro Arg Asn Val Gly Gly As - #p Leu Asp Pro Ser Ser      
         595          - #       600          - #       605                
  - - ATC CCC GAC AAG GAA CAG GCG ATC AGC GCC CT - #G CCG GAC TAC GCC AGC 
    1872                                                                  
 Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Le - #u Pro Asp Tyr Ala Ser      
     610              - #   615              - #   620                    
  - - CAG CCC GGC AAA CCG CCG CGC GAG GAC CTG AA - #G TAA                 
- #     1908                                                              
  Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Ly - #s                         
  625                 6 - #30                 6 - #35                     
   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                              
   - -      (i) SEQUENCE CHARACTERISTICS:                                 
            (A) LENGTH: 635 amino - #acids                                
            (B) TYPE: amino acid                                          
            (C) STRANDEDNESS: single                                      
            (D) TOPOLOGY: linear                                          
   - -     (ii) MOLECULE TYPE: protein                                    
   - -      (v) FRAGMENT TYPE: internal                                   
   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                        
   - - Met Glu His Trp Ser Tyr Trp Leu Arg Pro Gl - #y Glu Ala Gly Gly    
Gly                                                                       
   1               5  - #                10  - #                15        
  - - Gly Ser Gly Gly Gly Gly Ser Gly Gln Ala Ph - #e Asp Leu Trp Asn Glu 
             20      - #            25      - #            30             
  - - Cys Ala Lys Ala Cys Val Leu Asp Leu Lys As - #p Gly Val Arg Ser Ser 
         35          - #        40          - #        45                 
  - - Arg Met Ser Val Asp Pro Ala Ile Ala Asp Th - #r Asn Gly Gln Gly Val 
     50              - #    55              - #    60                     
  - - Leu His Tyr Ser Met Val Leu Glu Gly Gly As - #n Asp Ala Leu Glu Leu 
 65                  - #70                  - #75                  - #80  
  - - Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser As - #p Gly Leu Thr Ile Arg 
                 85  - #                90  - #                95         
  - - Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Le - #u Arg Tyr Ser Tyr Thr 
             100      - #           105      - #           110            
  - - Arg Gln Ala Arg Gly Arg Trp Ser Leu Asn Tr - #p Leu Val Pro Ile Gly 
         115          - #       120          - #       125                
  - - His Glu Lys Pro Ser Asn Ile Lys Val Phe Il - #e His Glu Leu Asn Ala 
     130              - #   135              - #   140                    
  - - Gly Asn Gln Leu Ser His Met Ser Pro Ile Ty - #r Thr Ile Glu Met Gly 
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Al - #a Thr Phe Phe Val    
Arg                                                                       
                  165  - #               170  - #               175       
  - - Ala His Glu Ser Asn Glu Met Gln Pro Thr Le - #u Ala Ile Ser His Ala 
             180      - #           185      - #           190            
  - - Gly Val Ser Val Val Met Ala Gln Asn Gln Pr - #o Arg Arg Glu Lys Arg 
         195          - #       200          - #       205                
  - - Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cy - #s Leu Leu Asp Pro Leu 
     210              - #   215              - #   220                    
  - - Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Ar - #g Cys Asn Leu Asp Asp 
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Al - #a Gly Asn Pro Ala    
Lys                                                                       
                  245  - #               250  - #               255       
  - - His Asp Leu Asp Ile Lys Pro Thr Val Ile Se - #r Glu Glu Leu Glu Phe 
             260      - #           265      - #           270            
  - - Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Al - #a His Gln Ala Cys His 
         275          - #       280          - #       285                
  - - Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gl - #n Pro Arg Gly Trp Glu 
     290              - #   295              - #   300                    
  - - Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Ar - #g Leu Val Ala Leu Tyr 
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - Leu Ala Ala Arg Leu Ser Trp Asn Gln Val As - #p Gln Val Ile Arg    
Asn                                                                       
                  325  - #               330  - #               335       
  - - Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Le - #u Gly Glu Ala Ile Arg 
             340      - #           345      - #           350            
  - - Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Th - #r Leu Ala Ala Ala Glu 
         355          - #       360          - #       365                
  - - Ser Glu Arg Phe Val Arg Gln Gly Thr Gly As - #n Asp Glu Ala Gly Ala 
     370              - #   375              - #   380                    
  - - Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pr - #o Val Ala Ala Gly Glu 
 385                 3 - #90                 3 - #95                 4 -  
#00                                                                       
   - - Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Le - #u Leu Glu Ala Asn    
Tyr                                                                       
                  405  - #               410  - #               415       
  - - Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gl - #y Asp Val Ser Phe Ser 
             420      - #           425      - #           430            
  - - Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Ar - #g Leu Leu Gln Ala His 
         435          - #       440          - #       445                
  - - Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Va - #l Gly Tyr His Gly Thr 
     450              - #   455              - #   460                    
  - - Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gl - #y Gly Val Arg Ala Arg 
 465                 4 - #70                 4 - #75                 4 -  
#80                                                                       
   - - Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Ph - #e Tyr Ile Ala Gly    
Asp                                                                       
                  485  - #               490  - #               495       
  - - Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gl - #n Glu Pro Asp Ala Arg 
             500      - #           505      - #           510            
  - - Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Va - #l Tyr Val Pro Arg Ser 
         515          - #       520          - #       525                
  - - Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Th - #r Leu Ala Ala Pro Glu 
     530              - #   535              - #   540                    
  - - Ala Ala Gly Glu Val Glu Arg Leu Ile Gly Hi - #s Pro Leu Pro Leu Arg 
 545                 5 - #50                 5 - #55                 5 -  
#60                                                                       
   - - Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gl - #y Gly Arg Leu Glu    
Thr                                                                       
                  565  - #               570  - #               575       
  - - Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Va - #l Val Ile Pro Ser Ala 
             580      - #           585      - #           590            
  - - Ile Pro Thr Asp Pro Arg Asn Val Gly Gly As - #p Leu Asp Pro Ser Ser 
         595          - #       600          - #       605                
  - - Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Le - #u Pro Asp Tyr Ala Ser 
     610              - #   615              - #   620                    
  - - Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Ly - #s                     
 625                 6 - #30                 6 - #35                      
  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 1191 base - #pairs                                 
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (ix) FEATURE:                                                   
           (A) NAME/KEY: Coding Se - #quence                              
           (B) LOCATION: 1...1188                                         
           (D) OTHER INFORMATION:                                         
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                         
  - - ATG GAG CAC TGG TCC TAT TGG CTG CGC CCT GG - #A GAA GCT GGA GGA GGA 
      48                                                                  
 Met Glu His Trp Ser Tyr Trp Leu Arg Pro Gl - #y Glu Ala Gly Gly Gly      
  1               5  - #                10  - #                15         
  - - GGA TCC GGA GGA GGA GGA TCC GGT CAA GCT TT - #T GTT AAC GCC CAT ATG 
      96                                                                  
 Gly Ser Gly Gly Gly Gly Ser Gly Gln Ala Ph - #e Val Asn Ala His Met      
             20      - #            25      - #            30             
  - - GCC GAA GAG GGC GGC AGC CTG GCC GCG CTG AC - #C GCG CAC CAG GCT TGC 
     144                                                                  
 Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Th - #r Ala His Gln Ala Cys      
         35          - #        40          - #        45                 
  - - CAC CTG CCG CTG GAG ACT TTC ACC CGT CAT CG - #C CAG CCG CGC GGC TGG 
     192                                                                  
 His Leu Pro Leu Glu Thr Phe Thr Arg His Ar - #g Gln Pro Arg Gly Trp      
     50              - #    55              - #    60                     
  - - GAA CAA CTG GAG CAG TGC GGC TAT CCG GTG CA - #G CGG CTG GTC GCC CTC 
     240                                                                  
 Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gl - #n Arg Leu Val Ala Leu      
 65                  - #70                  - #75                  - #80  
  - - TAC CTG GCG GCG CGG CTG TCG TGG AAC CAG GT - #C GAC CAG GTG ATC CGC 
     288                                                                  
 Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Va - #l Asp Gln Val Ile Arg      
                 85  - #                90  - #                95         
  - - AAC GCC CTG GCC AGC CCC GGC AGC GGC GGC GA - #C CTG GGC GAA GCG ATC 
     336                                                                  
 Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly As - #p Leu Gly Glu Ala Ile      
             100      - #           105      - #           110            
  - - CGC GAG CAG CCG GAG CAG GCC CGT CTG GCC CT - #G ACC CTG GCC GCC GCC 
     384                                                                  
 Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Le - #u Thr Leu Ala Ala Ala      
         115          - #       120          - #       125                
  - - GAG AGC GAG CGC TTC GTC CGG CAG GGC ACC GG - #C AAC GAC GAG GCC GGC 
     432                                                                  
 Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gl - #y Asn Asp Glu Ala Gly      
     130              - #   135              - #   140                    
  - - GCG GCC AAC GCC GAC GTG GTG AGC CTG ACC TG - #C CCG GTC GCC GCC GGT 
     480                                                                  
 Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cy - #s Pro Val Ala Ala Gly      
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - GAA TGC GCG GGC CCG GCG GAC AGC GGC GAC GC - #C CTG CTG GAG CGC    
AAC      528                                                              
  Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Al - #a Leu Leu Glu Arg Asn     
                 165  - #               170  - #               175        
  - - TAT CCC ACT GGC GCG GAG TTC CTC GGC GAC GG - #C GGC GAC GTC AGC TTC 
     576                                                                  
 Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gl - #y Gly Asp Val Ser Phe      
             180      - #           185      - #           190            
  - - AGC ACC CGC GGC ACG CAG AAC TGG ACG GTG GA - #G CGG CTG CTC CAG GCG 
     624                                                                  
 Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Gl - #u Arg Leu Leu Gln Ala      
         195          - #       200          - #       205                
  - - CAC CGC CAA CTG GAG GAG CGC GGC TAT GTG TT - #C GTC GGC TAC CAC GGC 
     672                                                                  
 His Arg Gln Leu Glu Glu Arg Gly Tyr Val Ph - #e Val Gly Tyr His Gly      
     210              - #   215              - #   220                    
  - - ACC TTC CTC GAA GCG GCG CAA AGC ATC GTC TT - #C GGC GGG GTG CGC GCG 
     720                                                                  
 Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Ph - #e Gly Gly Val Arg Ala      
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - CGC AGC CAG GAC CTC GAC GCG ATC TGG CGC GG - #T TTC TAT ATC GCC    
GGC      768                                                              
  Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gl - #y Phe Tyr Ile Ala Gly     
                 245  - #               250  - #               255        
  - - GAT CCG GCG CTG GCC TAC GGC TAC GCC CAG GA - #C CAG GAA CCC GAC GCA 
     816                                                                  
 Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln As - #p Gln Glu Pro Asp Ala      
             260      - #           265      - #           270            
  - - CGC GGC CGG ATC CGC AAC GGT GCC CTG CTG CG - #G GTC TAT GTG CCG CGC 
     864                                                                  
 Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Ar - #g Val Tyr Val Pro Arg      
         275          - #       280          - #       285                
  - - TCG AGC CTG CCG GGC TTC TAC CGC ACC AGC CT - #G ACC CTG GCC GCG CCG 
     912                                                                  
 Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Le - #u Thr Leu Ala Ala Pro      
     290              - #   295              - #   300                    
  - - GAG GCG GCG GGC GAG GTC GAA CGG CTG ATC GG - #C CAT CCG CTG CCG CTG 
     960                                                                  
 Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gl - #y His Pro Leu Pro Leu      
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - CGC CTG GAC GCC ATC ACC GGC CCC GAG GAG GA - #A GGC GGG CGC CTG    
GAG     1008                                                              
  Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Gl - #u Gly Gly Arg Leu Glu     
                 325  - #               330  - #               335        
  - - ACC ATT CTC GGC TGG CCG CTG GCC GAG CGC AC - #C GTG GTG ATT CCC TCG 
    1056                                                                  
 Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Th - #r Val Val Ile Pro Ser      
             340      - #           345      - #           350            
  - - GCG ATC CCC ACC GAC CCG CGC AAC GTC GGC GG - #C GAC CTC GAC CCG TCC 
    1104                                                                  
 Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gl - #y Asp Leu Asp Pro Ser      
         355          - #       360          - #       365                
  - - AGC ATC CCC GAC AAG GAA CAG GCG ATC AGC GC - #C CTG CCG GAC TAC GCC 
    1152                                                                  
 Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Al - #a Leu Pro Asp Tyr Ala      
     370              - #   375              - #   380                    
  - - AGC CAG CCC GGC AAA CCG CCG CGC GAG GAC CT - #G AAG TAA             
 - #   1191                                                               
 Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Le - #u Lys                      
 385                 3 - #90                 3 - #95                      
  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 396 amino - #acids                                 
           (B) TYPE: amino acid                                           
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (ii) MOLECULE TYPE: protein                                     
  - -      (v) FRAGMENT TYPE: internal                                    
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                         
  - - Met Glu His Trp Ser Tyr Trp Leu Arg Pro Gl - #y Glu Ala Gly Gly Gly 
  1               5  - #                10  - #                15         
  - - Gly Ser Gly Gly Gly Gly Ser Gly Gln Ala Ph - #e Val Asn Ala His Met 
             20      - #            25      - #            30             
  - - Ala Glu Glu Gly Gly Ser Leu Ala Ala Leu Th - #r Ala His Gln Ala Cys 
         35          - #        40          - #        45                 
  - - His Leu Pro Leu Glu Thr Phe Thr Arg His Ar - #g Gln Pro Arg Gly Trp 
     50              - #    55              - #    60                     
  - - Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gl - #n Arg Leu Val Ala Leu 
 65                  - #70                  - #75                  - #80  
  - - Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Va - #l Asp Gln Val Ile Arg 
                 85  - #                90  - #                95         
  - - Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly As - #p Leu Gly Glu Ala Ile 
             100      - #           105      - #           110            
  - - Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Le - #u Thr Leu Ala Ala Ala 
         115          - #       120          - #       125                
  - - Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gl - #y Asn Asp Glu Ala Gly 
     130              - #   135              - #   140                    
  - - Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cy - #s Pro Val Ala Ala Gly 
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Al - #a Leu Leu Glu Arg    
Asn                                                                       
                  165  - #               170  - #               175       
  - - Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gl - #y Gly Asp Val Ser Phe 
             180      - #           185      - #           190            
  - - Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Gl - #u Arg Leu Leu Gln Ala 
         195          - #       200          - #       205                
  - - His Arg Gln Leu Glu Glu Arg Gly Tyr Val Ph - #e Val Gly Tyr His Gly 
     210              - #   215              - #   220                    
  - - Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Ph - #e Gly Gly Val Arg Ala 
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gl - #y Phe Tyr Ile Ala    
Gly                                                                       
                  245  - #               250  - #               255       
  - - Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln As - #p Gln Glu Pro Asp Ala 
             260      - #           265      - #           270            
  - - Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Ar - #g Val Tyr Val Pro Arg 
         275          - #       280          - #       285                
  - - Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Le - #u Thr Leu Ala Ala Pro 
     290              - #   295              - #   300                    
  - - Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gl - #y His Pro Leu Pro Leu 
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Gl - #u Gly Gly Arg Leu    
Glu                                                                       
                  325  - #               330  - #               335       
  - - Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Th - #r Val Val Ile Pro Ser 
             340      - #           345      - #           350            
  - - Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gl - #y Asp Leu Asp Pro Ser 
         355          - #       360          - #       365                
  - - Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Al - #a Leu Pro Asp Tyr Ala 
     370              - #   375              - #   380                    
  - - Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Le - #u Lys                 
 385                 3 - #90                 3 - #95                      
  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 5 amino - #acids                                   
           (B) TYPE: amino acid                                           
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                         
  - - Gly Gly Gly Gly Ser                                                 
  1               5                                                       
  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 14 amino - #acids                                  
           (B) TYPE: amino acid                                           
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                         
  - - Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Se - #r Lys Val Asp         
  1               5  - #                10                                
  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 18 amino - #acids                                  
           (B) TYPE: amino acid                                           
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                         
  - - Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Le - #u Ala Gln Phe Arg Ser 
  1               5  - #                10  - #                15         
  - - Leu Asp                                                             
__________________________________________________________________________

Claims (37)

What is claimed is:
1. A method for detecting a tumor cell in a biological specimen, comprising contacting the biological specimen with a chimeric toxin with comprises gonadotropin releasing hormone and Pseudomonas exotoxin A, and detecting chimeric toxin-bound cells in the specimen.
2. The method of claim 1 in which biological specimen contains adenocarcinoma cells.
3. The method of claim 2 in which the adenocarcinoma cells are selected from a group consisting of colon adenocarcinoma, breast adenocarcinoma, lung adenocarcinoma, overian adenocarcinoma, endometrial adenocarcinoma, kidney adenocarcinoma, liver adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, cervical adenocarcinoma, gall bladder adenocarcinoma and pancreatic adenocarcinoma.
4. The method of claim 1 in which the Pseudomonas exotoxin is a full-length toxin.
5. The method of claim 1 in which the Pseudomonas exotoxin contains only domains II and III of a full-length toxin.
6. The method of claim 1 in which the chimeric toxin comprises the amino acid sequence as shown in SEQ ID NO:2.
7. The method of claim 6 in which the chimeric toxin is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:1.
8. The method of claim 1 in which the chimeric toxin comprises the amino acid sequence of SEQ ID NO:4.
9. The method of claim 8 in which the chimeric toxin is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:3.
10. The method of claim 1 in which the Pseudomonas exotoxin is altered to be non-cytotoxic.
11. The method of claim 10 in which the Pseudomonas exotoxin is altered to be non-cytotoxic by deleting an amino acid residue.
12. The method of claim 1 in which the chimeric toxin comprises the amino acid sequence as shown in SEQ ID NO:2 wherein amino acid residue #575 is deleted.
13. The method of claim 12 in which the chimeric toxin is encoded by a polynucleotide which comprises the nucleotide sequence as shown as SEQ ID NO:1 wherein nucleotides #1822-1824 are deleted.
14. The method of claim 1 in which the chimeric toxin comprises the amino acid sequence as shown in SEQ ID NO:4 wherein amino acid residue #336 is deleted.
15. The method of claim 14 in which the chimeric toxin is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:3 wherein nucleotides #1105-1107 are deleted.
16. The method of claim 1 in which the chimeric toxin is conjugated to a detectable label.
17. The method of claim 16 in which the detectable label is a radioisotope, a fluorescent dye, an enzyme, an ultrasonic probe or a NMR probe.
18. The method of claim 1 in which the biological specimen is a biopsy specimen.
19. The method of claim 1 in which the biological specimen is a bodily fluid.
20. The method of claim 19 in which the bodily fluid is whole blood.
21. The method of claim 19 in which the bodily fluid is pleural effusion fluid.
22. The method of claim 19 in which the bodily fluid is urine.
23. A method of detecting a tumor cell in a human subject, comprising administering to the subject a chimeric toxin which comprises gonadotropin releasing hormone and Pseudomonas exotoxin A, and detecting chimeric toxin-bound cells in the subject.
24. The method of claim 23 in which the subject has adenocarcinoma.
25. The method of claim 24 in which the adenocarcinoma is selected from a group consisting of colon adenocarcinoma, breast adenocarcinoma, lung adenocarcinoma, overian adenocarcinoma, endometrial adenocarcinoma, kidney adenocarcinoma, liver adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, cervical adenocarcinoma, gall bladder adenocarcinoma and pancreatic adenocarcinoma.
26. The method of claim 23 in which the Pseudomonas exotoxin is altered to be non-cytotoxic.
27. The method of claim 26 in which the Pseudomonas exotoxin is altered to be non-cytotoxic by deleting an amino acid residue.
28. The method of claim 23 in which the chimeric toxin comprises the amino acid sequence as shown in SEQ ID NO:2 wherein amino acid residue #575 is deleted.
29. The method of claim 28 in which the chimeric toxin is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:1 wherein nucleotides #1822-1824 are deleted.
30. The method of claim 23 in which the chimeric toxin comprises the amino acid sequence as shown in SEQ ID NO:4 wherein amino acid residue #336 is deleted.
31. The method of claim 30 in which the chimeric toxin is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:3 wherein nucleotides #1105-1107 are deleted.
32. The method of claim 23 in which the chimeric toxin is conjugated to a detectable label.
33. The method of claim 32 in which the detectable label is a radioisotope, a fluorescent dye, an enzyme, an ultrasonic probe or a NMR probe.
34. A chimeric toxin comprising gonadotropin releasing hormone and Pseudomonas exotoxin A which comprises the amino acid sequence as shown in SEQ ID NO:2 wherein the amino acid residue #575 is deleted.
35. The chimeric toxin of claim 34 which is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:1 wherein nucleotides #1822-1824 are deleted.
36. A chimeric toxin comprising gonadotropin releasing hormone and Pseudomonas exotoxin A which comprises the amino acid sequence as shown in SEQ ID NO:4 wherein the amino acid residue #336 is deleted.
37. The chimeric toxin of claim 36 which is encoded by a polynucleotide which comprises the nucleotide sequence as shown in SEQ ID NO:3 wherein nucleotides #1105-1107 are deleted.
US09/046,992 1998-03-24 1998-03-24 Methods of cancer diagnosis using a chimeric toxin Expired - Fee Related US6140066A (en)

Priority Applications (10)

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US09/046,992 US6140066A (en) 1998-03-24 1998-03-24 Methods of cancer diagnosis using a chimeric toxin
AT99910652T ATE412756T1 (en) 1998-03-24 1999-03-24 METHOD FOR DIAGNOSING CANCER USING A CHIMERIC TOXIN
DE69939819T DE69939819D1 (en) 1998-03-24 1999-03-24 METHOD FOR CANCER DIAGNOSIS BY MEANS OF A CHIMERIC TOXIN
EP99910652A EP1066390B1 (en) 1998-03-24 1999-03-24 Methods of cancer diagnosis using a chimeric toxin
CN99806580A CN1303438A (en) 1998-03-24 1999-03-24 Method of cancer diagnosis using chimeric toxin
AU29551/99A AU758839B2 (en) 1998-03-24 1999-03-24 Methods of cancer diagnosis using a chimeric toxin
NZ507294A NZ507294A (en) 1998-03-24 1999-03-24 Methods of cancer diagnosis using a chimeric toxin comprising GnRH and PE
CA002323786A CA2323786A1 (en) 1998-03-24 1999-03-24 Methods of cancer diagnosis using a chimeric toxin
PCT/IL1999/000166 WO1999049059A2 (en) 1998-03-24 1999-03-24 Methods of cancer diagnosis using a chimeric toxin
IL13865599A IL138655A0 (en) 1998-03-24 1999-03-24 Methods of cancer diagnosis using a chimeric toxin

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CA (1) CA2323786A1 (en)
DE (1) DE69939819D1 (en)
IL (1) IL138655A0 (en)
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US6838553B1 (en) * 1999-10-05 2005-01-04 Academia Sinica Peptide repeat immunogens
US20050079171A1 (en) * 1997-07-11 2005-04-14 The Government Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services Pseudomonas exotoxin A-like chimeric immunogens for eliciting a secretory IgA-mediated immune response
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US7704506B2 (en) 1995-12-18 2010-04-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Fcε-PE chimeric protein for targeted treatment of allergy responses a method for its production and pharmaceutical compositions containing the same
US20090082549A1 (en) * 1995-12-18 2009-03-26 Yissum Research Development Company Of The Hebrew University Of Jerusalem FcE-PE chimeric protein for targeted treatment of allergy responses a method for its production and pharmaceutical compositions containing the same
US20090181894A1 (en) * 1996-06-04 2009-07-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Chimeric toxins for targeted therapy
US20050256049A1 (en) * 1996-06-04 2005-11-17 Yissum Res. Dev. Co. Of The Hebrew U Of Jerusalem Chimeric toxins for targeted therapy
US20030166503A1 (en) * 1997-07-04 2003-09-04 Van Groeninghen Johannes C. J. Method for recognizing and determining GnRH receptors and use of GnRH agonist for decreasing the replication of malignant cells bearing GnRH receptors of tumors orginating in the nervous system and/or meninges and/or of Kaposi sarcoma
US8962558B2 (en) 1997-07-04 2015-02-24 Johannes C. van Groeninghen Methods for reducing GnRH-positive tumor cell proliferation using the GnRH antagonist IN3
US7695722B2 (en) 1997-07-04 2010-04-13 Van Groeninghen Johannes C Methods for reducing GNRH-positive tumor cell replication
US8092809B2 (en) 1997-07-11 2012-01-10 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pseudomonas exotoxin A-like chimeric immunogens
US20050079171A1 (en) * 1997-07-11 2005-04-14 The Government Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services Pseudomonas exotoxin A-like chimeric immunogens for eliciting a secretory IgA-mediated immune response
US7314632B1 (en) 1997-07-11 2008-01-01 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pseudomonas exotoxin A-like chimeric immunogens
US20090269368A1 (en) * 1997-07-11 2009-10-29 The Gov. Of The Usa As Represented By The Secretary Of The Dept.Of Health And Human Services Pseudomonas exotoxin a-like chimeric immunogens
US20050079154A1 (en) * 1998-03-02 2005-04-14 Yissum Research And Development Company Chimeric proteins with cell-targeting specificity and apoptosis-inducing activities
US20090317358A1 (en) * 1998-03-02 2009-12-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem Chimeric proteins with cell-targeting specificity and apoptosis-inducing activities
US20080255339A1 (en) * 1998-03-02 2008-10-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Chimeric proteins with cell-targeting specificity and apoptosis-inducing activities
US6838553B1 (en) * 1999-10-05 2005-01-04 Academia Sinica Peptide repeat immunogens
US20100247518A1 (en) * 2001-02-12 2010-09-30 Rosenblum Michael G Modified proteins, designer toxins, and methods of making thereof
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US20080292544A1 (en) * 2001-02-12 2008-11-27 Rosenblum Michael G Modified Proteins, Designer Toxins, and Methods of Making Thereof
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US20090010917A1 (en) * 2001-07-17 2009-01-08 Rosenblum Michael G Therapeutic Agents Comprising Pro-Apoptotic Proteins
US20030086919A1 (en) * 2001-07-17 2003-05-08 Rosenblum Michael G. Therapeutic agents comprising pro-apoptotic proteins
US20110002910A1 (en) * 2001-07-17 2011-01-06 Rosenblum Michael G Therapeutic agents comprising pro-apoptotic proteins
US8043831B2 (en) 2001-07-17 2011-10-25 Research Development Foundation Therapeutic agents comprising pro-apoptotic proteins
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US20040013691A1 (en) * 2002-06-12 2004-01-22 Rosenblum Michael G. Immunotoxin as a therapeutic agent and uses thereof
US20060171919A1 (en) * 2005-02-01 2006-08-03 Research Development Foundation Targeted polypeptides
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US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria

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ATE412756T1 (en) 2008-11-15
IL138655A0 (en) 2001-10-31
WO1999049059A2 (en) 1999-09-30
EP1066390A2 (en) 2001-01-10
NZ507294A (en) 2003-05-30
AU758839B2 (en) 2003-04-03
WO1999049059A3 (en) 1999-12-02
DE69939819D1 (en) 2008-12-11
CA2323786A1 (en) 1999-09-30
EP1066390B1 (en) 2008-10-29
AU2955199A (en) 1999-10-18

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